Many RMM technologies have been developed over the last 20-30 years. Most of these have been marketed mainly in the clinical sector, and to a lesser extent in the food manufacturing and water microbiology sectors. Nevertheless, some have specific pharmaceutical applications and others have been developed solely for the pharmaceutical industry.
Growth based RMM technologies differ from conventional culture methods in that they rely on the detection of biochemical or physiological growth indicators rather than visible growth. This generally allows much more rapid detection, but often requires a short enrichment stage before micro-organisms can be detected, especially in samples containing low levels of contamination. Examples of growth-based RMM include:
Adenosine triphosphate (ATP) bioluminescence is a well established rapid method for assessing contamination levels in pharmaceutical products and raw materials. It utilises a specific substrate and enzyme combination, luciferin/luciferase, to break down microbial ATP from growing cells and produce visible light, which can be measured using a luminometer. The amount of light is related to the number of microbial cells present. The main drawback is that non-microbial ATP is also detected.
Several commercial systems have been developed for a range of pharmaceutical test applications, especially for filterable samples where non-microbial ATP in the sample is less of a concern. Where low numbers of organisms (<100 CFU) are present, an enrichment step preceding the ATP bioluminescence assay is typically required. A step designed to reduce non-microbial ATP levels prior to the release of microbial ATP may also be required, usually using an enzyme and surfactant preparation, such as the Celsis LuminAse™ Biologic reagent. ATP-bioluminescence tests typically require 24-48 hours to complete including enrichment.
Examples include the Milliflex® Rapid system from Millipore, the Pallchek™ Rapid Microbiology System and the Celsis RapiScreen™ Biologics system. Celsis also markets an ‘enhanced’ ATP bioluminescence assay, AMPiScreen™, which uses the enzyme proprietary enzyme technology to increase the quantity of microbial ATP produced and reduce detection times by 25-50%.
Colorimetric growth detection
Colorimetric growth detection methods rely on a colour change being produced in a growth medium as a result of microbial metabolism during growth, often as a result of CO2 production. Clearly the range of organisms that can be detected by this method is limited by variations in metabolism, but systems able to detect most aerobes and acid-producing types, such as lactobacilli and yeasts, are available.
Two examples of commercial colorimetric assay system is the BacT/ALERT® 3D system from bioMerieux, originally designed for clinical applications, but also suitable for testing some pharmaceutical materials. And the BioLumix
system designed for the pharmaceutical market and capable of detecting the organisms specified in USP <61> and <62>. This is semi-automated and employs sensitive colour detection and analysis technology to produce a result within 24-48 hours.
All living cells auto fluoresce under blue light and this can be used to detect microbial colonies growing on a solid surface long before they are visible to the naked eye. This technique is particularly useful for filterable samples, where a membrane filter can be incubated on a conventional nutrient medium and scanned using highly sensitive imaging systems to detect microcolonies several days earlier than using traditional colony counting methods. Auto fluorescence detection has been commercialized by Rapid Micro Biosystems as The Growth Direct™ System, which uses a large area CCD imaging system without magnification to detect developing microcolonies in approximately half the time of the conventional culture method. The system has the advantage that it is non-destructive and mirrors the compendial method, thus is straightforward to validate. The system is also fully automated from sample prep onward.
Viability based RMM technologies do not rely on microbial growth to detect contamination, but instead use cell labelling techniques to detect and quantify viable microorganisms. This approach has the potential to detect a wide range of organisms, including yeasts and moulds, within a few minutes, but where very low levels of contamination (<10 CFU/ml) are present an enrichment step may be required. Several commercial products are available, all employing similar technology.
Molecular biology-based microbial detection systems have been making rapid progress in the clinical and food microbiology sectors and are now also being developed for pharmaceutical applications. The most commonly used technique to date is the polymerase chain reaction (PCR), which targets and amplifies specific sections of microbial nucleic acids to provide a highly specific detection technique, which, in the case of real-time PCR where amplification and detection are simultaneous, can produce results in a few hours. Many commercial PCR systems target specific microbial species, such as Salmonella, but there are also products aimed at the pharmaceutical industry, which are able to detect a much wider range of contaminants.
Molecular based microbial identification systems are also available for pharmaceutical applications and are described in more detail in Test Method Guide to Molecular Techniques for Microbial Identification and Typing
Endotoxins are lipopolysaccharide contaminants derived from the cell walls of Gram-negative bacteria. Endotoxin detection is an important function for pharmaceutical microbiology laboratories and is usually done using the long-established Limulus Amoebocyte Lysate (LAL) assay. Rapid semi-automated systems using sophisticated electronics have been developed for endotoxin testing and are claimed to be capable of real-time tests for PAT applications.
An example of a rapid endotoxin test system is the Endosafe® detection system available from Charles River Laboratories International. Endosafe uses a quantitative LAL-based chromogenic assay method together with a sensitive spectrophotometer and dedicated detection software. It comes as a multi-cartridge automated system that can analyse five samples in about 15 minutes, and as a portable hand-held version.
Endotoxin testing is decribed in more detail in
Test Method Guide to Endotoxin Detection Methods for Pharmaceuticals and Medical Devices
Rapid air monitoring
Environmental monitoring is another key requirement in pharmaceutical manufacturing facilities and rapid detection of airborne contamination in clean areas is particularly important. Most microbiological air sampling systems rely on conventional culture technology and so cannot give rapid results, but several instruments have been developed that can speed up this process.
For example, variations on impinger air sampler designs, such as the Coriolis®μ cyclonic air sampler (validated according to ISO 14698) made by Bertin Technologies (France), and the SAS-PCR sampler from pbi International, use a sterile liquid media, rather than the more common agar plate or strip; it allows subsequent detection of microbial contamination by rapid and specific molecular methods for identification and/or quantification. More rapid still are BioVigilant® Systems Instantaneous Microbial Detection (IMD) instruments, which sample the air continuously and use a technique called Mie scattering to measure particle size in combination with laser-induced intrinsic fluorescence to indicate biological activity. IMD technology is one of very few methods that can produce results in real time. It can alert operators immediately when contamination is detected. But other techniques are needed to subsequently identify the contaminants and the source of contamination.
In the past, difficulties with validation have proved to be a serious obstacle for the implementation of RMM in pharmaceutical manufacturing. But this has been changing recently and both the US and European Pharmacopoeias now include chapters on the validation of alternative microbiological methods (USP 1223 & EP 5.1.6). The Parenteral Drug Association (PDA) has also published guidance as Technical Report TR-33. The regulatory authorities now offer more encouragement for manufacturers to validate alternative methods and several rapid product release test methods have already been approved by the FDA.